1. Field of the Invention
The present invention relates generally to processes for preparing body fluid samples for their subsequent analysis using sample component separation techniques. More particularly, the present invention involves methods for removing low molecular weight components from urine samples, thus providing samples which are free of compounds which interfere with the electrophoretic separation and detection of higher molecular weight species.
2. Description of Relevant Art
Proteins present in mammalian body fluids such as whole blood, serum, plasma, cerebrospinal fluids, tears, sweat, saliva and urine are useful as indicators of the presence or absence or certain disease states. Thus, the ability to identify and quantitate a variety of proteins in body fluid clinical samples can provide diagnosticians a great deal of information leading to the diagnosis of a variety of diseases.
For example, patients inflicted with kidney disease will excrete urine containing albumin and other serum proteins which are typically absent in the urine of healthy individuals. Additionally, the urine of myeloma patients is known to have free light chain gamma globulins, proteins not normally excreted in the urine of patients free of myeloma. Accordingly, techniques for identifying and quantitating these and other protein components of clinical urine samples can provide indicators of abnormal conditions such as kidney disease and the presence of myeloma in patients.
A number of techniques involving the analysis of proteins found in body fluids are known. These range from wet chemistry methods which simply indicate the presence or absence of proteins to relatively complex methods involving the separation, identification, and quantitation of proteins which may be present at very low concentrations. These methods typically involve separating the fluid components using electrophoretic or chromatographic techniques followed by detecting the separated proteins. Typically, the detection methods involve directly analyzing the separated proteins by measuring their uv absorption using a detection wavelength at which the protein has a relatively high absorbance. Other optical detection methods include those which involve labelling the sample proteins with fluorescent labels or chemiluminescent labels and then detecting the separated proteins using fluorescent or chemiluminescent detectors. All of these methods have the advantage of providing qualitative as well as quantitative information when utilized in connection with standard curves generated from known proteins of known concentration.
Since mammalian proteins are charged molecules, mixtures of proteins can be subjected to electrophoretic separation techniques resulting in the separation of the protein components. In particular, recently developed capillary electrophoresis techniques provide efficient and rapid separations of small concentrations of charged species, and have become the method of choice for the rapid separation and analysis of charged components of clinical samples.
In general, capillary gel electrophoresis involves introducing a sample into a capillary column and applying an electric field across the column. The electric field causes the charged sample components to move within the gel filled column with the direction and speed of the movement being determined by the electrophoretic mobility of each charged component. The electrophoretic mobility in turn is dependent upon the mass of each of the sample components with those components having greater mobility travelling faster than those with slower mobility. This results in the sample components being resolved into discrete zones in the capillary column.
Another form of capillary electrophoresis or "open tube" CE is similar to the above-described gel capillary electrophoresis except that the column is filled with an electrically conductive buffer solution. Upon applying an electric field to the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. Under the influence of the electrical potential caused by the electric field, the bulk solution must flow toward the cathode in order to maintain electroneutrality. This electroendosmotic flow provides a fixed velocity component which drives both neutral species and ionic species towards the cathode.
Typically capillary gel electrophoresis and open-tube CE utilize an on-line detector such as a uv absorbance detector or other optically based detector to monitor separations and provide quantitative and qualitative data relating to the separated components. Proteins inherently absorb in the ultraviolet spectrum at 214 nm and 280 nm, making uv detectors the detector of choice because the proteins do not require special labelling. One problem associated with using uv detection methods is the presence of low molecular weight sample components which may be present at relatively high concentrations in clinical samples. Typically, these low molecular weight components are not the analyte of interest, but absorb at the preferred monitoring wavelength, which for proteins is 214 nm. Frequently, these lower molecular weight components will co-migrate with the proteins of interest, thus causing separation and detection problems. Even when the lower molecular weight components do not co-migrate they can be present at such high relative concentrations that the lower concentration components of interest are not detected. Thus, the lower molecular weight components can substantially interfere with the detection of the higher molecular weight analytes of interest which are typically present at much lower concentrations.
Attempts to overcome problems associated with interfering sample components generally involve procedures directed toward removing the unwanted components from the sample prior to performing the separations. These procedures include subjecting the sample to dialysis to separate low molecular weight components, solvent extraction techniques to partition low molecular components, precipitation, and centrifugation. In some cases, the sample is concentrated in order to increase the concentration of analytes which are known to be present at very low concentrations and not detectable in the presence of smaller molecular weight interfering components. These procedures are tedious and labor intensive, add cost and time to the analysis process and are generally considered unacceptable by clinical practitioners.
The practice of separating low molecular weight components from clinical samples is associated primarily with procedures involving the assay of urine for Bence Jones (BJ) protein in possible myeloma patients and the analysis of certain serum proteins in proteinuria patients. The amount of Bence Jones protein in urine varies from patient to patient and can be difficult to detect in the presence of low molecular weight interfering sample components. Membrane dialysis has been effective in removing the interfering components. However, this method is cumbersome, slow and requires large volumes of buffer solutions.
Accordingly, it would be desirable to provide methods for pretreating body fluid clinical samples in order to remove clinical sample components which interfere with the analysis of the clinical samples. Furthermore, it would be desirable to provide methods for analyzing body fluid samples for certain analytes while eliminating the effects of the presence of interfering components. More particularly, there is a need to provide methods for analyzing patient urine samples for low concentrations of Bence Jones protein and other proteins indicative of certain disease states.